Investigation Title Transcription profiling by array of brains from Africanized and European honey bees of different ages Comment[Submitted Name] Transcription profiling of brains from Africanized and European honey bees of different ages Experimental Design environmental_history strain_line loop_design dye_swap_design transcription profiling by array Experimental Design Term Source REF mo mo The MGED Ontology EFO Comment[ArrayExpressReleaseDate] 2010-12-31 Comment[SecondaryAccession] Comment[AEMIAMESCORE] 4 Comment[ArrayExpressAccession] E-TABM-953 Comment[MAGETAB TimeStamp_Version] 2010-10-14 12:10:03 Last Changed Rev: 14677 Experimental Factor Name Strain Age Experimental Factor Type strain_or_line age Experimental Factor Term Source REF Person Last Name Alaux Person First Name Cedric Person Mid Initials Person Email cedric.alaux@avignon.inra.fr Person Phone Person Fax Person Address Institute for Genomic Biology, 1206 West Gregory Drive, Urbana, IL 61801 USA Person Affiliation Institute for Genomic Biology, University of Illinois at Urbana Champaign Person Roles submitter Person Roles Term Source REF The MGED Ontology Quality Control Type biological_replicate dye_swap_quality_control Quality Control Term Source REF The MGED Ontology The MGED Ontology Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2010-12-31 PubMed ID 22691501 Publication DOI 10.1073/pnas.1205283109 Publication Author List Seth A. Ament; Charles A. Blatti; Cedric Alaux; Marsha M. Wheeler; Amy L. Toth; Yves Le Conte; Greg J. Hunt; Ernesto Guzmán-Novoa; Gloria DeGrandi-Hoffman; Jose Luis Uribe-Rubio; Gro V. Amdam; Robert E. Page; Sandra L. Rodriguez-Zas; Gene E. Robinson; Saurabh Sinha Publication Title New meta-analysis tools reveal common transcriptional regulatory basis for multiple determinants of behavior Publication Status Publication Status Term Source REF Experiment Description Expression profiling of honey bees brains as a function of genotype (AHB/EHB) and age Protocol Name P-TABM-4969 P-TABM-2964 P-TABM-2967 P-TABM-2966 P-MEXP-1269 Protocol Type grow nucleic_acid_extraction labeling hybridization image_acquisition Protocol Description 4 and 14 day-old AHB and EHB individuals reared in their own colonies Brains were partially lyophilized as in Grozinger CM, Sharabash NM, Whitfield CW, Robinson GE. Proc Natl Acad Sci U S A 2003, 100:14519-14525., with the following changes: dissections were carried out in an ethanol bath kept on dry ice. Individual brains were homogenized in 500μl of Trizol (Invitrogen Life Technologies). The mixture was incubated for 5 min and then 100μl of water and 100μl of Chloroform were added and allowed to incubate for 3 min. The solution was centrifuged at 12,000 g (4°C) for 15 min. The aqueous phase was mixed with an equal volume of 70% ethanol and transferred into a Qiagen RNeasy column. RNA extraction was carried out as indicated in the Qiagen RNeasy kit for total RNA with on-column DNase I treatment (Qiagen, Valencia, CA). RNA (500ng) was amplified with the Amino Allyl MessageAmp™ II aRNA Amplifcation kit (Ambion, Austin, TX), according to kit instructions. in vitro transcription proceeded with an incubation time of 4 h at 37° C. aaRNA sample were dried in SpeedVac and resuspend in 4.5 μl Coupling buffer (0.1M carbonate buffer pH 9). 4.5 μl Cy-dye esters in DMSO (GE/ Amersham; these solutions are prepared by resuspending one tube of each Cy-dye in 55 μl DMSO) was added to each sample. Samples were incubated at room temp in the dark for 1 hour with shaking. 4.5 μl 4 M hydroxylamine (Sigma) was added to each sample. Samples were incubated at room temperature for 15 min in the dark. Cy3 and Cy5 reactions were combined. 3 μl Nuclease-free Water was added to each sample to bring the volume to 30 μl. 105 μl of aRNA Binding Buffer was added to each aRNA sample. 75 μl 100% ethanol was added and pipetted 3 times to mix. Samples were applied on the"labeled aRNA Filter Cartridge" and centrifuge 10K x g for 1 minute. Discard flow-through and replace filter cartridge in tube. 500 μl Wash Buffer was added and centrifuge 1 minute at 10K x g. Discard flow-through and replace filter cartridge in tube. Centrifuge 1.5 minutes at 10K x g to dry column. Filter cartridge was transfered to a "Labeled aRNA Elution Tube".20 μl 55C water Nuclease-free Water was added to filter cartridge. Incubate at room temperature for 2 minutes. Centrifuge 1.5 minute at 10K x g. Repeat the water elution to yield a final volume of ~ 40 ul labeled aaRNA. Slides were passed quickly through steam and placed in a UV linker at 6000 x 100μJ/cm2. Before pre-hybridization, slides were plunged in 0.2% SDS and immediately shaken vigorously for 2 min. They were then washed twice in distilled water, transferred to 95% ethanol for 15 sec, and dried at 2000 rpm for 3 min. For hybridization, slides were incubated at 42o C in a Coplin jar for ~1h30, then washed in distilled water twice and isopraponol and dried at 2000 rpm for 3 min. Samples were incubated at 95-100° C for 3 min and then kept at 55° C until applied to the microarray slides. 75-80 ul of sample was applied on the slides and slides incubated for 18 h at 42°C. Slides were scanned using an Axon 4000B scanner. Images were analyzed with GenePix software. Spot finding by software was manually checked for all microarrays. Protocol Parameters Protocol Hardware Axon GenePix 4000B scanning hardware Protocol Software GenePix Protocol Contact Protocol Term Source REF SDRF File E-TABM-953.sdrf.txt Term Source Name mo NCI_thesaurus ncbitax EFO The MGED Ontology ArrayExpress The MGED Ontology mo EFO Term Source File http://mged.sourceforge.net/ontologies/MGEDontology.php ncithesaurus.obo.alt http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://www.ebi.ac.uk/efo/ http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version